Method for assaying the antioxidant capacity of a sample

ABSTRACT

A kit for assaying the antioxidant capacity of a sample, the kit including an extraction solution including a solubility enhancing compound to be added to a sample for extracting antioxidants present in the sample; and a fluorescent probe to be added to the extract.

RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 10/077,018, filed on Feb. 15, 2002, incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to an improved method for assaying theantioxidant capacity of a sample.

BACKGROUND OF THE INVENTION

Molecules are composed of atoms bonded together. This bonding process isaccomplished by the sharing of electrons. When two atoms come togetherand their electrons pair up, a bond is created. Generally, only twoelectrons can exist in one bond. Paired electrons are quite stable andalmost all electrons in the human body exist in a paired state. However,when a bond is broken, the electrons can either stay together or splitup. If the electrons stay together, both electrons go to one of theatoms and none go to the other atom. In this case the molecularfragments are called ions, which are electrically charged and typicallynot harmful to humans or other animals. For example, sodium chloride,NaCl, can split up into a sodium cation (Na⁺) and a chloride anion(Cl⁻). But, if the electrons split up when a bond is broken, oneelectron will go to each atom, creating two molecules with unpairedelectrons, called free radicals. The unpaired electrons of these freeradicals are highly energetic and unstable and seek out other electronswith which to pair with. As a result, free radicals steal electrons fromother molecules. The process of stealing electrons from other electronpairs is what makes free radicals dangerous because it causes oxidation,or loss of electrons, to the molecule it attacks, leaving an unstable,highly energetic molecule. Since most electrons exist in a paired state,free radicals often end up reacting with paired electrons and createstill more free radicals. Only when a free radical pairs up with anotherfree radical is the free radical terminated.

Antioxidants, or free radical scavengers, function by offering easyelectron targets for free radicals. In absorbing a free radical,antioxidants “trap”, or de-energize and stabilize the lone free-radicalelectron and make it stable enough not be harmful.

As such, antioxidants provide a defense against free radicals whichcause cell oxidation in humans and other animals. Presently, there isoverwhelming evidence to indicate that free radicals cause oxidativedamage to lipids, proteins and nucleic acids. Antioxidants can play animportant role in the prevention of a number of diseases includingcancer, heart, vascular, and neurogenitive diseases. See Oxygen-RadicalAbsorbance Capacity for Antioxidants, Cao, G., Free Radical Biol. Med.Vol. 14, (1993), incorporated herein in its entirety by this reference.

Many foods contain substantial quantities of antioxidants. The need toeffectively measure the antioxidant capacity of such foods is ofsignificant importance to people who are trying to prevent diseasescaused by free radicals, to manufacturers of foods alleged to containhigh antioxidant capacities, and to the scientific community. Moreover,in the medical community, measuring the antioxidant capacity of bloodand serum can be useful in prevention of disease. Accordingly, manyfood, vitamin and supplement suppliers seek to test the antioxidantcapacity of their various products. In addition, biological samples areoften tested to determine their antioxidant capacity.

In 1993, the Oxygen Radical Absorbance Capacity (ORAC) assay wasdeveloped to test the antioxidant capacity of a given sample. SeeOxygen-Radical Absorbance Capacity for Antioxidants cited above. And, in1998, an automated device, the Roche COBAS FARA II analyzer, was placedon the market to test samples according to the ORAC assay. Moreover,significant research has been performed to determine the antioxidantcapacity of samples using the ORAC assay. See, e.g. Oxygen RadicalAbsorbance Capacity (ORAC) and Phenolic and Anthocyanin Concentrationsin Fruit and Leaf Tissues of Highbush Blueberry, Ehlenfeldt, M. andPrior, R., J. Agric. Food Chem., 49, pp. 2222-2227 (2001); In Vivo TotalAntioxidant Capacity: Comparison of Different Analytical Methods, Prior,R. and Cao, G., Free Radical Biol. Med., Vol. 27, Nos. 11/12, pp.1173-1181 (1999); Total Antioxidant Capacity of Fruits, Wang, H., Cao,G., Prior, R., J. Agric. Food Chem., 44, pp. 701-705 (1996); andAntioxidant Capacity of Tea and Common Vegetables, Cao, G., Sofic, E.,and Prior, R., J. Agric. Food Chem., 44, pp. 3426-3431 (1996), allincorporated herein in their entirety by this reference.

Since about 1998, the inventors hereof have used the COBAS FARA II totest various samples according to the ORAC assay. In performing the ORACassay numerous times, the inventors hereof detected and herein delineatesolutions to numerous problems associated with the conventional ORACassay.

In accordance with the published ORAC assay, a sample such as freshfruit, blood serum, or an additive or supplement in powder form isprepared for extraction and extracted first in water and then inacetone. A protein based fluorescent probe, namely B-phycoerythrin(B-PE) is then added to the extract. A standard, having high antioxidantcapacity, such as diluted grape seed extract (GSE) or Trolox, a watersoluble analog of vitamin E, is added to the extract to provide acomparison of the antioxidant capacity of the sample to the standard.The extract, the standard, and a blank sample are then loaded into theCOBAS FARA II device and an initial fluorescence emission of the probeis taken. Next, AAPH, (2,2′-azobis (2-amidino-propane) dihydrochloride),which generates free radicals upon heating, is added to the extract ofthe sample and the standard and fluorescence emission readings are takenuntil a zero value is reached for the extract of the sample. To measurethe protective effect of an antioxidant using the ORAC assay, the areaunder the fluorescence decay curve (AUC) of the sample is calculated andcompared to that of the blank in which no antioxidant is present.

One problem with this prior art assay is that samples including highlevels of lipid soluble antioxidants are not correctly rated because oftheir insolubility in aqueous media. Further, samples including bothlipid soluble antioxidants and water-soluble antioxidants are notcorrectly rated.

Another problem with the prior art ORAC assay is that the probe used wasB-PE. This protein probe was found to interact with the sample inadverse ways and generated false low readings. Moreover, because B-PE ismanufactured from a microorganism, it was found to vary in purity andcomposition from lot to lot. In addition, B-PE is highly photosensitivewhich is a severe drawback when fluorescence intensity decay is used inthe assay in that B-PE requires special handling.

Another problem with the prior art ORAC assay is that since only onestandard is used, calculating the antioxidant capacity of the samplebased on the fluorescence intensity decay of the probe in both thesample and the standard incorrectly assumes that a direct ratio betweenthe antioxidant capacity of the standard and the sample could be made.This, however, is not true.

Still another problem with the prior art ORAC assay is that percloricacid was added to biological samples to separate proteins from thesample. The inventors hereof discovered that percloric acid, itself astrong oxidizing agent, yielded false low antioxidant capacity readings.

Finally, the prior art ORAC procedure involved a long dwell time of upto 75 minutes.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide an improvedmethod of assaying the antioxidant capacity of a sample.

It is a further object of this invention to provide such a method whichaccurately measures the antioxidant capacity of a sample even if it hasa high level of lipid soluble antioxidants.

It is a further object of this invention to provide such a method whichaccurately measures the antioxidant capacity of a sample having bothhigh level of lipid soluble antioxidants and water soluble antioxidants.

It is a further object of this invention to a provide a probe which doesnot interact with the sample.

It is a further object of this invention to provide such a method usinga probe which is more stable.

It is a further object of this invention to provide such a method inwhich the probe is consistent in purity from lot to lot.

It is a further object of this invention to provide such a method usinga probe which is not photosensitive.

It is a further object of this invention to provide such a method inwhich a plurality of standards are used to accurately measure theantioxidant capacity of a sample.

It is a further object of this invention to provide such a method whichuses a non-chemical means to remove proteins from a sample.

It is a further object of this invention to provide such method which isa more time efficient method for assaying the antioxidant capacity of asample.

This invention results from the realization that an improved method forassaying the antioxidant capacity of a sample, in the preferredembodiment, can be achieved by preparing an extraction solution whichincludes a solubility enhancing compound; by using a unique non-proteinprobe which does not interact with the sample, and which is consistentlypure, and which is not photosensitive; by employing a plurality ofstandards which correctly account for the ratio between the antioxidantcapacity of the standard and the sample; by using non-chemical means toremove the proteins thereby eliminating any interaction with the sample;and by increasing the concentration of AAPH to decrease the dwell time.

This invention features a method of assaying the antioxidant capacity ofa sample, the method including preparing an extraction solutionincluding a solubility enhancing compound, adding the sample to theextraction solution, extracting the antioxidants present in the sample,adding a fluorescent probe to the extract, detecting the fluorescenceintensity decay of the probe in the presence of the sample over time,and calculating the antioxidant capacity of the sample based on thefluorescence intensity decay of the probe in the presence of the sample.

The method of assaying the antioxidant capacity in accordance with thisinvention may include solubility enhancing solution having a highpolarity solvent and a low polarity solvent wherein the solubilityenhancing compound enhances the solubility of lipid soluble antioxidantspresent in the sample in the high polarity solvent. Ideally, the highpolarity solvent is water, and the low polarity solvent is selected fromacetone, butane, methanol, acetonitrile and ethanol. In one embodiment,the solubility enhancing compound is cyclodextrine and derivativesthereof. The amount of the high polarity solvent may be equal to orapproximately equal to the amount of low polarity solvent. Thesolubility enhancing compound may be 1% to 40% of the solution.

The method of assaying the antioxidant capacity in accordance with thisinvention may also include a non-protein probe which is a hydrogen atomdonor probe. Preferably, the hydrogen atom donor probe is fluoresceinand derivatives thereof.

This invention also features a method of assaying the antioxidantcapacity, the method including adding the probe to a plurality ofstandards each having a known antioxidant capacity, and detecting thefluorescence intensity decay of the probe in the presence of eachstandard over time. Preferably, the calculation step includes comparingthe fluorescence intensity decay of the probe in the presence of samplewith the fluorescence intensity decay of the probe in the presence ofeach standard. In a preferred embodiment there are four standards, withthe concentration of the standards ranges from 10 μM to 100 μM. Ideally,each standard is Trolox. The preparation step may include removing anyproteins present in the sample by using non-chemical means to remove theproteins, such as an ultra-filtration technique. A free radicalgenerator precursor is added to the probe/extract mixture, such as AAPHabove 4 mM. The preferred concentration of AAPH is 12.8 mM.

This invention also features a method of assaying the antioxidantcapacity in which a microplate fluorescence reader is used to detect theintensity decay of the probe in the presence of the sample over time.Ideally, the microplate fluorescence reader is a FL600 microplatefluorescence reader. An automatic pipetting system may be used to dilutethe sample, such as a Precision 2000 automatic pipetting system. In apreferred embodiment, the sample is automatically diluted with a buffersolution to a concentration of the sample to buffer in the range of 1:40to 1:320. Ideally, the automatic pipetting system adds the fluorescentprobe to the sample. The automatic pipetting system may also add thefluorescent probe to the plurality of standards. The automatic pipettingsystem may additionally add the free radical generator precursor to theprobe/extract mixture.

This invention also features a method of assaying the antioxidantcapacity of a sample including preparing an extraction solution, addingthe sample to the solution, extracting the antioxidants present in thesample, adding a non-protein probe to the extract, detecting thefluorescence intensity decay of the non-protein probe in the presence ofthe sample over time, and calculating the antioxidant capacity of thesample based on the fluorescence intensity decay of the non-proteinprobe in the presence of the sample.

This invention further features a method of assaying the antioxidantcapacity, the method including preparing an extraction solutionincluding a solubility enhancing compound, adding the sample to theextraction solution, extracting the antioxidants present in the sample,adding a fluorescent probe to the extract, adding a free radicalgenerator precursor to the extractor solution, detecting thefluorescence intensity decay of the probe in the presence of the sampleover time, and calculating the antioxidant capacity of the sample basedon the fluorescence intensity decay of the probe in the presence of thesample.

This invention further features a kit for assaying the antioxidantcapacity of a sample including an extraction solution including asolubility enhancing compound to be added to a sample for extractingantioxidants present in the sample, and a fluorescent probe to be addedto the extract. The extraction solution includes a high polarity solventsuch as water and a low polarity solvent selected from the acetone,butane, methanol, acetonitrile and ethanol. The kit may include asolubility enhancing compound, such as cyclodextrin and derivativesthereof. The amount of the high polarity solvent may be equal to orapproximately equal to the amount of low polarity solvent. Ideally, thesolubility enhancing compound is 1% to 40% of the solution. The kit mayinclude a non-protein probe which is a hydrogen atom donor probe, suchas fluorescein, and may also include a plurality of standards eachhaving a known antioxidant capacity so that the fluorescence intensitydecay of the probe in the presence of each standard over time can bedetected. There may be four standards, ranging from 10 μM to 100 μM.Ideally, the standard is Trolox. The kit may include adding a freeradical generator precursor to be added to the probe/extract mixture,such as AAPH. In one embodiment, the concentration of the precursor isabove 4 mM. Ideally, the concentration of the precursor is 12 mM.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other objects, features and advantages will occur to those skilled inthe art from the following description of a preferred embodiment and theaccompanying drawings, in which:

FIG. 1 is a schematic block diagram showing the primary steps associatedwith the preparation of a sample to be tested in accordance with thesubject invention;

FIG. 2 is schematic block diagram showing the primary steps associatedwith analyzing the antioxidant capacity of a sample using the COBAS FARAII in accordance with the subject invention;

FIGS. 3A-3C are schematic diagrams showing the primary steps associatedwith analyzing the antioxidant capacity of a sample using an FL600microplate fluorescence reader and a Precision 2000 automatic pipettingsystem;

FIG. 4 is a schematic diagram showing one configuration of the deckstation of the Precision 2000 automatic pipetting system in accordancewith the subject invention;

FIG. 5 is a schematic diagram of a multiple-well microplate located atone of the stations of a Precision 2000 automatic pipetting systemshowing one configuration of various samples, blank solutions, andstandard solutions, in accordance with the subject invention;

FIG. 6 is a schematic diagram of a multiple-well microplate located atanother station of the Precision 2000 automatic pipetting system showingone configuration of diluted samples, blank solutions, standardsolutions, and control solutions in accordance with the subjectinvention;

FIG. 7 is a graph showing the fluorescence intensity decay over time ofa sample being tested, a standard, and a blank for a typical ORAC assay;

FIG. 8 is a schematic block diagram showing the primary steps associatedwith the method for assaying the antioxidant capacity of a preparedsample in accordance with the subject invention;

FIG. 9 is schematic block diagram of illustrating the primary stepsassociated with another embodiment of the method for assaying theantioxidant capacity of a sample in accordance with the subjectinvention;

FIG. 10 is a depiction of the structure the non-protein probefluorescein used for assaying the antioxidant capacity of a sample inaccordance with the subject invention;

FIG. 11 is a graphical representation showing how there is nointeraction between the non-protein probe and the sample in accordancewith the subject invention;

FIG. 12 is a graphical representation showing the strong interactionbetween a protein probe B-PE and a sample;

FIG. 13 is graphical representation showing the fluorescent intensitydecay of four standards as used in one embodiment in accordance with thesubject invention;

FIG. 14 is a graphical representation showing the calculated net areaunder the curve versus the concentration of one standard compared to theplurality of standards in accordance with the subject invention; and

FIG. 15 is a schematic block diagram illustrating the primary stepsassociated with another embodiment of the method for assaying theantioxidant capacity of a sample in accordance with the subjectinvention.

DETAILED DESCRIPTION OF THE INVENTION

As explained in the Background of the Invention section above,antioxidants provide a defense against free radicals which causeoxidation in humans and other animals. Antioxidants can neutralize freeradicals and play an important role in the prevention of a number ofdiseases. Many foods contain substantial quantities of antioxidants andthe need to effectively measure the antioxidant capacity of such foodsis of significant importance to people who are trying to preventdiseases caused by free radicals, manufacturers of foods alleged tocontain high antioxidant capacities, and in the scientific community.Moreover, in the medical community, measuring the antioxidant capacityof blood and serum can be useful in the prevention of disease.

In accordance with the subject invention, samples for testing may bereceived either in powdered form or in coarse form. A sample is receivedin powdered form, step 10, FIG. 1. The sample is then dissolved in anacetone and water mixture, typically a 50% acetone and a 50% watermixture, step 12. If a coarse sample is received, step 14, it is groundin a machine mill to produce a fine powder, step 16. In either case,after the sample is dissolved in the acetone water mixture, the solutionis diluted with a buffer, step 18. However, if the sample is liquid,such as blood serum, or any other bodily fluid, as shown at step 20, itis directly diluted with a buffer as shown in step 18. The solution isthen shaken using an orbital shaker for approximately 1 hour, step 22.The extract solution is then centrifuged, step 24; membrane filtrationis performed to remove any proteins (e.g. blood or serum samples), step26; the extract is then centrifuged again, step 28; and the supernatantis removed for analysis, step 30. The sample is now ready for analysiswith the COBAS FARA II analyzer (Roche Diagnostic System Inc.,Branchburg, N.J.; emission filter 565 nm).

Using the COBAS FARA II analyzer, the sample is first pipetted into theCOBAS FARA II tube, step 32, FIG. 2; a buffer and main reagent is thenadded into the wells of the covette rotor of the COBAS FARA II, step 34.The rotor is then spun to mix the sample buffer and reagents, step 36;the solution is then incubated for 30 seconds, step 38. An initialfluorescence (f₀) is taken while the rotor is spinning, step 40; therotor is stopped from spinning and a sample of AAPH reagent is added tostart the oxidative reaction, step 42; and fluorescence decay readingsare taken every half second then every minute for thirty-five minutes(f₁, f₂, f₃ . . . ), step 44.

A FL600 microplate fluorescence reader (Bio-Tek Instruments, Inc.,Winooski, Vt.) may also be used sample analysis. Ideally the FL600microplate reader employs fluorescence filters with an excitationwavelength of 485±20 nm and an emission wavelength of 530±25 nm. TheFL600 microplate fluorescence reader is controlled by software, such asKC4 3.0 (reversion 29). Sample dilution is performed by an automatedliquid handler with a robotic multi-channel liquid handling system inwhich the samples are diluted in series. Preferably, a Precision 2000automatic pipetting system managed by software, such as Precision Powersoftware (version 1.0), (Bio-Tek Instruments, Inc., Winooski, Vt.) isemployed.

Automated sample preparation begins by placing two pipette racks 202 and204, FIG. 4 into two separate stations 206 and 208, respectively, of thePrecision 2000 automatic pipetting system, step 210, FIG. 3A. In oneexample, 250 μL 96-pipette racks used. Fluorescein is then dispensedinto first reagent holder 210, FIG. 4 located, in one example at station211, step 212, FIG. 3A. Preferably, 50 mL 8.16×10⁻⁵ mM fluorescein isplaced in first reagent holder 210. A buffer solution is dispensed intosecond reagent holder 212, step 214, FIG. 3A. Ideally, 50 ml 75 mMphosphate buffer (pH 7.4) is added to second reagent holder 212, FIG. 4.AAPH solution is added to third reagent holder 216, FIG. 4, as shown bystep 216, FIG. 3A. A first multiple-well plate 230, FIG. 4 is placed inthird separate station 232 of the Precision 2000, step 218, FIG. 3A.Preferably, a 96-well polypropylene plate with a maximum well volume of320 μL is used.

Samples are then manually added using a multi-channel pipette to twospaced columns 234 and 238 of first multiple-well plate 230, FIG. 5,indicated by step 232, FIG. 3A. In one example, eight samples arepipetted into first spaced column 234, FIG. 5 (wells A1-H1), and eightadditional samples are pipetted into second spaced column 238 (wellsA6-H6).

A blank solution is then dispensed into column 240, FIG. 5 of firstmultiple well plate 230, step 242, FIG. 3A. In one example, 200 μL of 75mM phosphate buffer (blank) is dispensed into column 240, FIG. 5 (wellsA11-H11).

A standard solution at various concentration is dispensed into separatecolumn 244, FIG. 5 of first multiple well plate 230, step 246, FIG. 3A.In one example, a Trolox standard solution is added to column 244, FIG.5 of first multiple well plate 230 (wells A12-H12). The variousconcentrations of Trolox standard solution in column 244, in oneexample, are be 6.25 μM (well A12), 12.5 μM (well B12), 25 μM (wellC12), 50 μM (well D12), 50 μM (well E12), 25 μM (well F12), 12.5 μM(well G12), and 6.25 μM (well H12).

In order to perform series dilution of the samples in column 234 and 238of first multiple well plate 230, FIG. 5, an automated pipetting systemis used, such as a Precision 2000 managed by dilution sequencingsoftware, such as Precision Power software (version 1.0). The samples intwo spaced columns 234 and 238 are diluted into plurality of discreetcolumns 241, 243, 245, and 247, and 248, 250, 252, and 254,respectively, step 314, FIG. 3A. Ideally, each column of diluted sampleadjacent to sample columns 234 and 238, FIG. 5 has a lower concentrationof sample than the next. In one example, the sample series dilution ofsamples in columns 234 and 238 performed by the Precision 2000controlled by the dilution sequencing software resulted in a dilution of1:40 (sample to buffer) in columns 241 (wells A2 to H2) and column 248(wells A7 to H7). Consecutive dilutions of 1:2, 1:2, and 1:2 areperformed by the Precision 2000 to the samples in columns 241-247 and248-254, respectively, by the Precision 2000. The result, in oneexample, is a series of diluted samples at ratios of 1:40, 1:80, 1:160,and 1:320, as shown in FIG. 5. Alternatively, any other desired lowerdilution can be obtained by performing a series of 1:4 or 1:8 dilutionsas after initial 1:40 dilution. Moreover, any other desired series ofdilutions can be produced as needed.

A second multiple well plate 274, FIG. 4 is placed into fourth separatestation 312 of the Precision 2000, step 272, FIG. 3B. Secondmultiple-well plate 274 is needed to provide for an automatedplate-to-plate transfer of the diluted samples from first multiple wellplate 230 to second multiple well plate 274. In one example, a second96-well polystyrene plate is placed in station E of the FL600 microplatereader. A fully automated plate-to-plate liquid transfer is programmedby the Precision Power software. The robotic liquid handling system ofthe Precision 2000 driven by the Precision Power software, transfersfluorescein from first reagent holder 210, FIG. 4, to all of the wellsof second multiple well plate 274 located in station 275 (e.g., StationE) of the Precision 2000, step 278, FIG. 3B. In one example, 150 μL offluorescein solution from reagent holder 210, FIG. 4 is transferred toall columns of first multiple well plate 274.

The blank solution in column 240 of first multiple well plate 230 istransferred to column 302 of second multiple well plate 274 using theautomated and programmed plate-to-plate liquid transfer system describedabove, step 304, FIG. 3B. Ideally, 25 μL of blank solution istransferred from column 240 (wells A11-H11) of first multiple well plate274, FIG. 5 to column 302 (wells A12-H12), FIG. 6 of second multiplewell plate 274.

The standard solution (e.g. Trolox) in column 244 of first multiple wellplate 230, FIG. 5, is transferred to column 282, FIG. 6, in secondmultiple well plate 274 via the plate-to-plate liquid transfer system,step 306, FIG. 3B.

The plurality of discrete adjacent columns of diluted samples (e.g.,columns 241-247 and 248-254, FIG. 5) in first multiple well plate 230are automatically transferred to a plurality of discrete adjacentcolumns in second multiple well plate 274 by the liquid transfer system,step 308, FIG. 3B. In one example, 25 μL of diluted sample solutionsfrom the adjacent columns 241, 243, 245, and 247 in first multiple wellplate 230, FIG. 5, are transferred to columns 284, 286, 288, and 290,respectively, of second multiple well plate 274. Similarly, 25 μL ofdiluted sample solutions from the adjacent columns 248, 250, 252, and254, FIG. 5, in first multiple well plate 230, are transferred tocolumns 292, 294, 296, and 298, respectively, of second multiple wellplate 274 by the Precision 2000 automatic pipetting system controlled bythe dilution sequencing software.

The control solution from third multiple well plate 312, FIG. 4, locatedin fifth separate station 312 (e.g., Station F) of the Precision 2000 istransferred to separate column 300, FIG. 6, in second multiple wellplate, step 310, FIG. 3B. In one example, 25 μL of 25 μM gallic acid istransferred from column 314 of third multiple well plate 312, FIG. 4, tocolumn 300 of second multiple-well plate 274, FIG. 6.

Second multiple well plate 274 is then immediately covered and incubatedin the preheated (37° C.) FL600 microplated fluorescence reader for tenminutes and provided with a three minute shaking, step 350, FIG. 3B.Second multiple well plate 274 is then transferred back to station 275(e.g., Station E) of the FL6000 microplate fluorescence reader, step354, FIG. 3C. AAPH is then transferred from third reagent holder 214,FIG. 4, to all of the columns of the second multiple well plate 274,FIG. 6, excluding columns 280 and 302 wherein the blank solutions arelocated, step 356, FIG. 3C. Hence, when the automated sample preparationis complete, the total volume for each well is ideally 200 μL.

Second multiple well plate 274 is then transferred to FL6000 microplatefluorescence reader and fluorescence is measured every minute forthirty-five minutes, step 358, FIG. 3C.

FIG. 6 shows the layout of a typical 96-well plate used for ameasurement with the FL600 microplate fluorescence reader. Although thesamples, standard, and blank solutions are located at specific columns,in FIGS. 5 and 6, and the pipette racks, multiple well plates andreagent holders are located at specific stations in the Precision 2000,this is not a necessary limitation of the invention as any configurationof the various columns and stations may be used.

After the fluorescent readings are taken, the ORAC values are calculatedby using a regression equation between standard concentration 62, FIG.7, and the net area under the B-PE decay curve of sample 60. ORAC valuesare typically expressed in μmole Trolox equivalent per liter or pergram.

In accordance with the subject invention the area under the curve (AUC)for the sample, standard, and blank are calculated as:AUC=0.5+f ₁ /f ₀ +f ₂ /f ₀ +f ₃ /f ₀ +f ₄ /f ₀ + . . . +f _(i) /f ₀  (1)Where f₀=initial fluorescence reading at 0 minute, and f_(i) is thefluorescence reading at time i. Typically, equation (1) is solved andthe data analyzed in an electronic spreadsheet such as Microsoft Excelor other similar products or computer programs. The AUC is calculatedfor sample 60, standard 62 and blank 64. The net AUC is obtained bysubtracting the AUC of blank 64 from sample 60. The relative ORAC valuefor sample 60, expressed in Trolox equivalents is calculated as:Relative ORAC value=[(AUC _(Sample)-AUC _(Blank))/(AUC _(Trolox)-UC_(Blank))]×(molarity of Trolox/molarity of sample)   (2)

Accordingly, the ORAC value yields the antioxidant capacity of thesample can be found.

One problem with the prior art ORAC assays is that oil solubleantioxidants are not correctly rated because of their insolubility inaqueous media. Further, most analytical instruments used for antioxidanttesting, such as the COBAS FARA II analyzer or FL600 microplatefluorescence reader are designed to handle only aqueous solutions. As aresult, these prior art ORAC assays are less ideal for lipid or oilsoluble samples. Moreover, most fat-soluble free radical precursorsgenerate free radicals only at high temperature (>70° C.) at which manyorganic solvents evaporate and create environmental hazards. Withoutknowing the actual effectiveness of oil soluble antioxidants, consumerscan be exposed to unsafe concentrations. In addition, samples includingboth lipid soluble antioxidants and water-soluble antioxidants are notcorrectly rated.

The applicants' unique method of assaying the antioxidant capacity of asample overcomes the problem associated with oil-based samples bypreparing an extraction solution which includes a solubility enhancingcompound. One such solubility enhancing compound is cyclodextrin and thederivatives thereof.

Cyclodextrins (CDs) are a group of naturally occurring cage moleculeswhich are built up from α-D-glucose units. Depending on the number ofglucose moieties in the ring (6, 7 or 8) they are named α-, β-, andγ-cyclodextrin. CDs are doughnut shaped and can bind a variety oforganic ‘guest’ compounds inside their apolar cavities in aqueoussolution. The main driving force for this binding is hydrophobicinteractions. There are also numerous compounds chemically derivatizedthrough the hydroxyl groups of CDs of which may be applied to thesubject invention.

Cyclodextrins (CDs) contain a relatively hydrophobic (fat-like) centralcavity and hydrophilic (water-like) outer surface. This property ofcyclodextrin has made it useful as a vehicle for enhancing thesolubility of fat-soluble compounds in an aqueous environment. See FattyAcid-Cyclodextrin Complexes: Properties and Applications, J. Incl. Phen.Mol. Recog. Chem., Szente, L., Szejtli, J. 16, 339-354 (1993);Introduction to Organic Chemistry, Streitwieser, A.; Heathcock, C. H.,429, Macmillan Publishing Co., Inc. New York (1976), each incorporatedherein in their entirety by this reference.

In one embodiment in accordance with the subject invention, the methodof assaying the antioxidant capacity of the sample, FIG. 8, includespreparing an extract solution including a solubility enhancing compound,step 46; adding the sample to the extraction solution, step 48;extracting the antioxidants present in the sample, step 50; adding afluorescence probe to the extract, step 52; detecting the fluorescenceintensity decay of the probe in the presence of the sample over time,step 54; and calculating the antioxidant capacity of the sample based onthe fluorescence intensity decay of the probe in the presence of thesample, step 56. Ideally, cyclodextrin is the solubility enhancingcompound and in one example, randomly methylated β-cyclodextrin (RMCD)is chosen as the solubility enhancing compound. Alternatively, anyderivatives of cyclodextrin may be used such as α, β, andγ-cyclodextrin.

As noted supra, lipid soluble antioxidants were not correctly ratedbecause of their insolubility in aqueous media. Moreover, samples withboth lipid and water-soluble antioxidants were not correctly rated.

To overcome the problem associated incorrect ORAC antioxidant ratingswhich result from samples which include both lipid soluble antioxidantsand water-soluble antioxidants, the applicants' unique method ofassaying the antioxidant capacity of a sample includes an extractionsolution which includes both a high-polarity solvent, such as water, alow-polarity solvent, such as acetone, butanone, methanol, acetonitrileand ethanol or other similar low polarity solvents, wherein thesolubility enhancing compound enhances the solubility of thelipid-soluble antioxidants which are present in the sample to be testedin the high-polarity solvent. The amount of the high-polarity solventmay be equal to or approximately equal to the amount of the low-polaritysolvent. In a preferred embodiment, the solubility enhancing compound is1% to 40% of the solution.

The applicants' unique method of assaying the antioxidant capacity ofthe sample which includes a solubility enhancing compound allows formore accurate representation of the actual antioxidant capacity of asample containing high-levels of lipid-soluble antioxidants as well aslipid soluble antioxidants with water-soluble antioxidants. Theapplicants' incorporation of cyclodextrin as a solubility enhancingcompound to the subject invention significantly increases the ORACvalues by enhancing the solubility of the fat-soluble compounds inaqueous environments. Moreover, by using a solubility enhancing compoundwith a high polarity and low polarity mixture, samples with lipidsoluble and water soluble antioxidants can accurately be tested.

Another problem associated with prior art ORAC assays is that a proteinprobe, such as B-PE is used. As discussed supra, the B-PE probeinteracts with the sample in adverse ways which can result in low falsereadings. This is because B-PE interacts with many polyphenoliccompounds resulting non-specific protein bindings. Further, the processof B-PE isolation from Porphyridium cruetum itself producesinconsistencies in purity which vary from lot to lot resulting invariable reactivity with free radicals. Moreover, B-PE is highlyphotosensitive, which is a significant drawback when fluorescenceintensity decay is used in an assay because the B-PE will requirespecial handling.

In sharp contrast, the applicants' preferred method of assaying theantioxidant capacity of the sample overcomes the problems associatedwith a protein based fluorescence probe by using a non-protein probe.The unique method of assaying the antioxidant capacity of a sample inaccordance with the subject invention includes preparing an extractsolution, step 70, FIG. 9; adding the sample to the solution, step 72;extracting the antioxidants present in the sample, step 74; adding anon-protein probe to the extract, step 76; detecting the fluorescenceintensity decay of the non-protein probe in the presence of the sampleover time, step 78; and calculating the antioxidant capacity of thesample based on the fluorescence intensity decay of the non-proteinprobe in the presence of the sample, step 80. In one example, thenon-protein probe is a hydrogen atom donor such as fluorescein,3′,6′-dihydroxyspiro[isobenzofuran-1(3H), 9′[9H]-xanthen]-3-one, asshown in FIG. 10. Other suitable derivatives of fluorescein may also beused.

The unique non-protein probe, when used in accordance with the subjectinvention, does not interact with the sample because fluorescein, unlikeB-PE, does not interact with polyphenolic compounds which can causenon-specific protein bindings. Moreover, since fluorescein is notmanufactured from a microorganism, it does not vary in purity from lotto lot like protein probes. Moreover, fluorescein is not photosensitiveand requires no special handling.

A comparison of the binding properties of fluorescein and B-PE probes isshown in FIGS. 11 and 12. As shown in FIG. 11, non-protein probefluorescein 100, when used in accordance with the subject invention,does not interact with Grape Seed Extract (GSE) 102, which is known topossess strong protein binding properties. In sharp contrast, the B-PEprotein probe 104, FIG. 12, strongly binds with the GSE samples 106,108, and 110.

Tables 1-3 below summarize the ORAC values of 16 chemicals and varioussamples measured using fluorescein probe in accordance with the subjectinvention and B-PE probes respectively. TABLE 1 Relative ORAC Values ofChemicals with Antioxidant Activities* Compounds ORAC_(FL) ORAC_(B-PE)Ratio* Caffeic Acid 4.37 ± 0.24 1.40 ± 0.09 3.12 Chlorogenic Acid 3.14 ±0.19 1.90 ± 0.12 1.65 Coumaric Acid 2.95 ± 0.24 1.45 ± 0.03 2.03Quercetrin 5.87 ± 0.49 2.70 ± 0.18 2.15 Genistein 5.93 ± 0.45  2.3 ±0.16 2.57 Glutathione 0.62 ± 0.02 0.32 ± 0.01 1.94 Rutin 4.28 ± 0.251.95 ± 0.21 2.19 Quercetin 4.38 ± 0.22 2.07 ± 0.05 2.11 Vitamin C 0.95 ±0.02 0.43 ± 0.03 2.21*ORAC values are expressed as relative Trolox equivalent calculatedbased on equation 2 (n > 3)

TABLE 2 ORAC_(FL) and ORAC_(PE) Values for Biological Fluids andBeverages* ORAC_(FL/) Sample ORAC_(FL) ORAC_(PE) ORAC_(PE) Urine 1542 ±178  926 ± 133 1.67 Whole Serum 7780 ± 467 3383 ± 278 2.30 Serum(protein free)   347 ± 5.63   186 ± 9.11 1.87 Blueberry Juice 23748 ±1555 7511 ± 683 3.16 Bilberry Juice 34659 ± 2069 12507 ± 893  2.77 GrapeJuice 31441 ± 1821 12124 ± 912  2.59 Raspberry Juice 54034 ± 2863 23056± 1800 2.34 Black Tea 17267 ± 441  8714 ± 213 1.89*ORAC values are expressed as micromole Trolox equivalent per liter (n >3)

TABLE 3 ORAC_(FL) and ORAC_(PE) of Various Natural Products Extracts*ORAC_(FL/) Sample ORAC_(FL) ORAC_(PE) ORAC_(B-PE) Bilberry 2646 ± 1901283 ± 144 2.06 Elderberry 2221 ± 164 1174 ± 182 1.89 Red Wine Extract6942 ± 669 2194 ± 105 3.16 Grape Seeds Extract A 11889 ± 234  3516 ± 1353.38 Grape Seeds Extract B 11681 ± 923  2989 ± 368 1.89*ORAC_(FL) and ORAC_(PE) values are expressed as micromole Troloxequivalents per gram (n > 3).

As shown above, the applicants' unique application of the non-proteinprobe fluorescein in accordance with the subject invention exhibitsdistinct advantage over the prior art protein probe B-PE. Because thereis no interaction between the non-protein probe fluorescein and thesample which would lower the measured ORAC value, the actual measuredORAC values of the antioxidant sample is significantly more accurate(higher) than prior art techniques which use B-PE.

Another problem with prior art ORAC assays is that only one standard wasused. When only one standard is used, calculating the antioxidantcapacity of the sample is based on the fluorescence intensity decay ofthe probe in both the sample and the standard which incorrectly assumesa direct ratio between the antioxidant capacity of the standard and thesample. That is, typical prior art useY(μM)=a (intercept)+bX(net area)   (3)to calculate the concentration of the sample and assume intercept (a) iszero, as shown by arrow 148, FIG. 14. For example, in the prior art, ifthe concentration of the standard is known (Y_(Standard)), and the AUCof the standard (X_(standard)) is the measured and calculated usingequation (1) above, and the AUC of the sample (X_(sample)) is measuredand calculated using equation (1), the desired concentration of thesample (Y_(sample)) is found by assuming (a) is zero and using the ratioof:Y _(Standard) /Y _(sample) =X _(standard) /X _(sample)   (4)and solving for Y_(sample). This however, as noted above, assumes theintercept of equation (3) is zero, when in fact it is not. Y_(sample) inthen used in equation (2) above and an erroneous ORAC value iscalculated.

The applicants' invention overcomes this inaccuracy and false assumptionby employing multiple standards at different known concentrations, hencesolving (a) of equation (3). Thereafter, the exact concentration of thesample can be found and applied to equation (2) above, yielding a moreaccurate ORAC value for the sample being tested.

In one preferred embodiment in accordance with the subject invention,the method for assaying the antioxidant capacity of a sample includesadding a probe which includes a plurality of standards having a knownantioxidant capacity, step 120, FIG. 15, which is supplementary to step52, FIG. 3. Thereafter, the fluorescence intensity decay of the probe isdetected in the presence of each standard, step 122; and a comparison ofthe fluorescence intensity of the probe in the presence of the samplewith the fluorescence intensity decay of the probe in the presence ofeach standard is performed, step 124. As shown in FIG. 13, fourstandards are used in addition to a blank standard. In one example,standards 124, 126, 128, and 130 and blank 132 are used. Preferably, thestandards 124, 126, 128, and 130 are Trolox at concentrations of 100 μM,50 μM, 25 μM and 12.5 μM, respectively. FIG. 10 illustrates thefluorescence decay curves of standards 124-130, as well as blank 132.These AUC of sample 134, standards 124-130, and blank 132 is calculatedusing equation (1) above. However, in accordance with the subjectinvention, standards 124, 126, 128, and 130 are applied to equation (3)and the exact value of intercept (a) is calculated. Thereafter, theexact concentration of sample 134 can be calculated and applied toequation (2) above. The result is a significantly more accurate ORACvalue because the assumption that intercept (a) of equation (3) is zerois not made as shown by arrow 150, FIG. 14. Table 4 below summarizes thetrolox calibration curve including the coalition coefficient (R²), slope(b) and intercept (a) of 9 runs of calculating the exact intercept ofthe standard in accordance with the subject invention. TABLE 4 Summaryof Trolox Calibration Curve [Y (μM) = a + bX(net area)] Run No. R² Slope(b) Intercept (a) 1 0.9994 2.5368 −2.174 2 0.9993 2.7390 −4.690 3 0.99812.6947 −5.109 4 0.9973 2.5291 −3.846 5 0.9928 2.2331  1.361 6 0.99782.8868 −3.788 7 0.9981 2.6288 −3.012 8 0.9987 2.5297 −2.589 Average0.9977 2.5846 −2.861 Acceptable Criteria ≧0.9900 NA NA

By using four standards instead of one, the exact correlation betweenthe antioxidant capacity of the sample and each standard can be made.Accordingly, by using the calculated intercept the correct concentrationof the sample can be found and a more accurate representation of theactual antioxidant capacity of the sample can be calculated.

Still another problem with the prior art ORAC assay is that percloricacid was added to biological samples to separate the proteins from thesamples. However, percloric acid itself is a strong oxidizing agent andyields false low antioxidant capacity readings.

In a preferred embodiment of the subject invention, a non-chemical meansis used to remove the proteins from the sample. The biological fluids tobe tested are filtered through a microcon filter tube with cut-offmolecular weight of 2500 g/mol. Ideally, 1.0 mL of the biological fluidto be tested is added to a microcon filter tube and centrifuged at 1400rpm for up to 1 hour at 4° C. The liquid at the bottom of the tube iscollected for ORAC analysis with the COBAS FARA II analyzer or FL600microplate fluorescence reader.

The applicants' unique method of using non-chemical means to remove thebiological sample prevents percloric acid, or any type of reagent, toact as and oxidizing agent which falsely lowers antioxidant capacityreadings of a sample. In accordance with the subject invention, theunique ultra filtration technique employed eliminates using any reactivereagents thereby eliminating any possible interaction of the reagentswith the sample. The result is a more accurate antioxidant capacityreading.

Finally, prior art ORAC procedures involve a long dwell time of up toseventy-five minutes. In a preferred embodiment of the subjectinvention, the method for assaying the antioxidant capacity of a sampleas set forth herein significantly reduces the dwell time by increasingthe concentration of the free radical generator precursor to theprobe/extract mixture. Ideally, the free radical generator precursor isAAPH at a concentration of the above 4 mM. Preferably, theconcentrations of the precursor is above 12.8 mM.

In yet another embodiment of the subject invention, a kit for assayingthe antioxidant capacity of a sample includes an extraction solutionincluding a solubility enhancing compound to be added to the sample forextracting antioxidants present in the sample and a fluorescent probeadded to the extract. The kit may include an extraction solutionincluding a high polarity solvent and a low polarity solvent: the highpolarity solvent may be water and the low polarity solvent may beacetone, butanone, methanol acetonitrile or ethanol, and may alsoinclude a solubility enhancing compound such as cyclodextrin and thederivatives thereof. The amount of the high polarity solvent may beequal to or approximately equal to the amount of low polarity solventand the solubility enhancing compound is typically 1% to 40% of thesolution. The kit may also include a non-protein probe which is ahydrogen atom donor probe such as fluorescein. In one example, the kitincludes a plurality of standards having a known antioxidant capacity sothat the fluorescent intensity decay of the probe in the presence ofeach standard over time can be detected. The four standards may rangefrom 10 μM to 100 μM. Each standard is preferably Trolox. A free radicalgenerator precursor, such as AAPH, may be added to the probe extractmixture. In one example, the concentration of the precursor is above 4mM, and preferably is 12 mM.

The applicants unique method for assaying the antioxidant capacity of asample includes a unique solubility enhancing compound which overcomethe problems associated with the insolubility of lipids in aqueousmedia. The unique non-protein probe accurately measures the ORAC valueof samples without interacting with the sample, is consistently purebecause it is not produced from a microorganism, and is notphotosensitive and hence requires no special handling. By using aplurality of standards, more accurate ORAC readings can be found becausethe calculations include the exact location of the zero intercept.Further, removal of proteins can be accomplished by without usingperchloric acid which interacts with the sample. Finally, dwell time ofthe ORAC assay is significantly reduced by increasing the concentrationof AAPH.

EXAMPLES

The following examples are meant to illustrate and not limit the presentinvention. Unless otherwise stated, all parts therein are by weight.

Example 1 Materials and Methods

Flavonoid compounds and β-phycoerythrin were purchased from Sigma (St.Louis, Mo.). Trolox, ascorbic acid and disodium fluorescein wereobtained from Aldrich (Milwaukee, Wis.). 2,2′-azobis (2-amidino-propane)dihydrochloride (AAPH) was purchased from Wako Chemicals USA (Richmond,Va.). Various analyzed samples were also obtained. ORAC analyses wereperformed on a COBAS FARA II analyzer (Roche Diagnostic System Inc.,Branchburg, N.J.; Excitation wavelength=493 nm and emission filter=515nm).

Ascorbic acid and flavonoids were directly dissolved in acetone/watermixture (50:50, v/v) and diluted with pH 7.4 phosphate buffer foranalysis. The solid samples were initially ground in a mechanical millto produce a fine power; then 0.5 grams were accurately weighed and 20mL of acetone/water (50:50, v/v) extraction solvent was added. Themixture was shaken at 400 RPM at room temperature on an orbital shakerfor one hour. The extracts were centrifuged at 14000 rpm for 15 min, andthe supernatant was ready for analysis after appropriate dilution withbuffer solution. For liquid samples, a 20 mL aliquot of sample wascentrifuged for 15 min and the supernatant was ready for analysis afterappropriate dilution. Blood plasma or serum was diluted 100 to 200 foldwith pH 7.4 phosphate buffer before analysis. To measure the ORAC innon-protein fraction, protein was removed using 0.5 N perchloric acid(1:1; v:v; plasma:acid), the samples were then centrifuged at 140,000×gfor 10 min at 4° C., and the supernatants were removed as the serumnonprotein fractions and appropriately diluted with pH 7.4 phosphatebuffer before the analysis.

Peroxyl radical scavenging assay. The COBAS FARA II was programmed touse a two-reagent system (Reaction Mode 3, P-I-SRI-A). The reaction modepipetted and transferred the sample (20 μL), phosphate buffer (5 μL,0.75 mM, pH 7.4), and main reagent (365 μL FL, 4.8×10⁻⁸ M) into the mainreagent wells of their respective cuvette rotor positions. With spinningof the rotor, the reagents are mixed and incubated for 30 s beforerecording the initial fluorescence (f₀). After the rotor stops spinning,a start reagent (SRI), 8 μL of APPH (0.64 M) plus 2 μL of the phosphatebuffer is pipetted into the appropriate start reagent well in thecurvette rotor. Next, the analyzer starts spinning, mixing of thesample/FL with AAPH reagent and the oxidative reaction starts. Hence,the sample makes up 5% of the reaction volume, and the finalconcentrations of FL and AAPH are 4.38×10⁻⁸ M and 1.28×10⁻² M,respectively. Between transfers, both sample and reagent transferpipettes are washed with clean solution to eliminate sample crosscontamination. Fluorescence readings are taken at 0.5 s and then everyminute thereafter (f₁, f₂, f₃ . . . ) for a duration of 30 min. If thefluorescence of the final reading has not declined by >95% from thefirst reading, the dilution of sample is adjusted accordingly and thesample is reanalyzed. To determine the maximum voltage for thephotomultiplier tube, the AAPH reagent is omitted and is replaced withbuffer, and the analysis is run for 10 min. FL and AAPH were prepared ina 0.75 mM phosphate buffer. FL working solution was routinelypreincubated in a waterbath at 37° C. for 15 min before loading into theCOBAS reagent rack. Phosphate buffer was used as a blank and Troloxconcentrations of 12.5, 25, 50, 100 μM were used as standards.

Example 2

Modified Assay for Lipophilic Antioxidants Using Solubility EnhancingRandomly Methylated Cyclodextrin

Chemicals and Apparatus. Cyclodextrin derivatives were supplied fromCyclodextrin Technologies Development, Inc. (Gainesville, Fla.).Fluorescein (FL) and Trolox were purchased from Aldrich (Milwaukee,Wis.). 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH) wasobtained from Wako Chemicals USA (Richmond, Va.). γ-Oryzanol waspurchased from TCI America (Portland, Oreg.); nutriene (tocotrienols)was obtained from Eastman Chemicals Company (Kingsport, Tenn.). Allother standards were commercially available form Sigma or Aldrich.Analyses were performed on a COBAS FARA II analyzer (Roche DiagnosticSystem Inc., Branchburg, N.J. using an excitation wavelength of 493 nmand an emission filter of 515 nm or alternatively, the sample may beanalyzed with a FL600 microplate fluorescence reader (Bio-TekInstruments, Inc., Winooski, Vt.) with fluorescence filters for anexcitation wavelength of 485±20 nm and a emission wavelength of 530±25nm. The plate reader is ideally controlled by software, such as KC4 3.0(reversion 29). Sample dilution is accomplished by a Precision 2000automatic pipetting system managed by software, such as precision powersoftware (version 1.0), Bio-Tek Instruments, Inc., Winooski, Vt. The96-well polystyrene microplates and covers may be purchased from VWRInternational, Inc. (Bridgeport, N.J.). High performance LiquidChromatography (HPLC) and mass spectroscopy conditions are the same asin reference (4).

Samples. Twelve seed oils and fourteen essential oils were obtained inhouse.

Sample Preparation. Approximately 0.5 g of sample was dissolved in 20 mLacetone. An aliquot of sample solution was appropriately diluted with 7%cyclodextrin solvent made in 50% acetone-water mixture (v/v) and wasshaken for 1 hour at room temperature on an orbital shaker. The samplesolution was ready for analysis after further dilution with 7%cyclodextrin solution.

The automated ORAC assay was carried out on a COBAS FARA IIspectrofluorometric centrifugal analyzer. In the final assay mixture(0.4 mL total volume), FL (4.38×10⁻⁸ M) was used as a target of freeradical attack and AAPH (1.28×10⁻² M) as a peroxyl radical generator.Trolox solutions (12.5, 25, 50, 100 μM) were used as control standards.The analyzer was programmed to record the fluorescence of FL everyminute after addition of AAPH. All measurements were expressed relativeto the initial reading. Final results were calculated using thedifferences of areas under the FL decay curves between the blank and asample. These results were expressed as micromole Trolox equivalents(TE) per gram or liter.

In accordance with the subject invention, ORAC values were measured forseveral plant seed oils and essential oils. The antioxidant activity ofthe plant seed oil varies greatly from several hundred μmolTE/liter(Olive Divanci) to over fifty thousand μmolTE/liter (Caraway). TheEssential oils have impressively high ORAC values per gram bases. Forexample Myrrh oil has over 4000 μmolTE/g, which is over three timeshigher than that of pure α-tocopherol (1162 μmolTE/g).

Using randomly methylated β-cyclodextrin as solubility enhancer avalidated assay for oxygen radical absorbance capacity of lipophilicantioxidants (ORAC_(lipo)) was established. The ORAC_(lipo) method isrobust, reliable and sensitive. The precision determined at eachconcentration level does not exceed 15% coefficient of variation. Thelimit of detection is 5 μM and the limit of quantitation is 12.5 μM.Steric hindrance around phenol groups may have negative effect on ORACvalues of tocopherols. The method has been applied in evaluation ofantioxidant activity of plant oils.

Example 3 Using a Plurality of Standards

Trolox concentrations of 20, 40, 75 μM were used as QC samples. Samplesand Trolox calibration solutions were always analyzed in duplicate in a“forward-then-reverse” order as follows: blank, 12.5 μM Trolox, 25 μMTrolox, 50 μM Trolox, 100 μM Trolox, QC, sample 1 . . . sample 1, QC,100 μM, 50 82 M, 25 μM, 12.5 μM, blank. This arrangement can correct forpossible errors due to the signal drifting associated with the differentpositions of the same sample. Nine samples can be tested in duplicate ineach analysis. The final ORAC values are calculated by using aregression equation between the Trolox concentration and the net areaunder the FL decay curve and are expressed as Trolox equivalents asmicromole per liter or per gram. The area under curve (AUC) iscalculated as shown above in equations (1) and (2).

Characterization of Fluerescein (FL) oxidized products. FL (4.38×10⁻⁷ M)was incubated at 37° C. for 20 min with AAPH (1.28×10⁻² M) at pH 7.4,and the reaction mixture was analyzed by LC/MS. Chromatographic analyseswere performed on an HP 1100 series HPLC equipped with anautosampler/injector, binary HPLC pump, column heater, diode arraydetector, fluorescence detector and HP ChemStation for data collectionand manipulation. Reverse phase separation was performed on a Zorbax C18column (2.1×150 mm, 3 μm) at 37° C. UV detection was recorded at 278 nmand for fluorescence detection, the excitation wavelength was 491 nm andthe emission wavelength was 515 nm. The binary mobile phase consisted of(A) water, acetonitrile, acetic acid (89:9:2) (B) water, andacetonitrile (20:80). The separation was performed using a lineargradient from 0% to 30% B in 30 min. The structural information wasobtained using an LCQ ion trap mass spectrometer (Thermoquest, San Jose,Calif.) equipped with an API chamber and an ESI source. The ionizationmode was negative mode, Aux gas and Sheath gas were set to 90 and 23units, respectively. An ionization reagent of 1.5 mM ammonium hydroxidewas added at a rate of 0.05 mL/min through a Tee device by using asecondary HPLC pump before the API chamber. Fluorescein disodium wasused as a standard for calibrating the system. As shown in FIG. 13, theHPLC output monitored at 278 nm and fluorescence at 493 nm excitationand 515 nm emissions of fluorescein and oxidized products in thepresence of AAPH.

The linear relationship between the net area and antioxidantconcentration was evaluated by the inventors hereof using Trolox, blacktea leaves, blueberry extracts and grape skin extracts at differentconcentrations. The results are summarized in Table 5 below, showing thenet areas corresponding to the different concentrations of black tealeaves, elderberry extract and grape seed extract and the calculatedORAC values. All analyzed samples in the various forms demonstrate agood linear relationship between the net area and concentration. Troloxwas used as a calibration standard. The limit of quantitation (LOQ) andthe limit of detection (LOD) are 12.5 and 5 μM, respectively. Anacceptable correlation of coefficient was≧0.99. TABLE 5 Net AreaCorresponding to Different Concentrations of Extracts from Tea,Blueberry and Grape Skins. Natural Products Conc. (mg/L) Net Area ORAC*Black Tea leaves 8 5.92 1586 16 10.81 1566 32 21.51 1629 BlueberryExtracts 5 5.73 2441 10 11.32 2635 20 22.98 2792 Grape Skin Extracts 1.28.34 15675 2.4 15.63 15521 4.8 29.89 14714*ORAC values are expressed as Trolox Equivalents per gram. The RSD foraverage value of each sample is less than 15%.

Example 4 High Throughput ORAC Assay Using Multi-Channel Liquid HandlingSystem with Microplate Fluorescence Reader

Chemicals and Apparatus. Trolox and fluorescein disodium were obtainedfrom Aldrich (Milwaukee, Wis.). 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH) was purchased from Wako Chemicals USA (Richmond,Va.). B-Phycoerythrin (B-PE) and 15 phenolic compounds were obtainedfrom Sigma Co. (St. Louis, Mo.). Coffee powder, rosemary extract,strawberry extract and grape juice were obtained in house. Plasma waswithdrawn from 3 volunteers at Brunswick Laboratories. A FL600microplate fluorescence reader (Bio-Tek Instruments, Inc., Winooski,Vt.) was used with fluorescence filters for an excitation wavelength of485±20 nm and an emission wavelength of 530±25 nm. The plate reader wascontrolled by software KC4 3.0 (reversion 29). Sample dilution wasaccomplished by a precision 2000 automatic pipetting system managed byprecision power software (version 1.0) (Bio-Tek Instruments, Inc.,Winooski, Vt.). The 96-well polystyrene microplates and the covers werepurchased from VWR International Inc (Bridgeport, N.J.). A COBAS FARA IIanalyzer (Roche Diagnostic System Inc., Branchburg, N.J.) was used for acomparison study.

Automated Sample Preparation. The automated sample preparation wasperformed using a Precision 2000. The layout of the deck of the Bio-TekPrecision 2000 is illustrated in FIG. 4. As shown, the 250 μL pipetteracks were placed at station A and D. Station B was the reagent vesselin which 50 mL 5.84×10⁻⁵ mM FL was placed in reagent holder 1 and 50 ml75 mM phosphate buffer (pH 7.4) was added in reagent holder 2. A 96-wellpolypropylene plate (maximum well volume 320 μL) was placed at station Cfor sample dilution. The initial addition of samples into the 96-wellplate at station C was done by manual mode using an 8-channel pipette.Briefly, 200 μL 75 mM phosphate buffer (blank) was dispensed into Column11 (wells A11-H11). The Trolox standard solution was added into column12 (wells A12-H12) as follows: 6.25 μM (A12), 12.5 μM (B12), 25 μM(C12), 50 μM (D12), 50 μM (E12), 25 μM (F12), 12.5 μM (G12), 6.25 μM(H12). Then 8 samples were pipetted into column 1 (wells A1-H1) andcolumn 6 (wells A6-H6), respectively. The sample series dilutionsequence was programmed and controlled by the precision power software(version 1.0). An initial 1:40 dilution was performed followed byconsecutive 1:2, 1:2 and 1:2 dilutions, this would give a series of1:40, 1:80, 1:160, and 1:320 dilutions. Any other desired lower dilutioncan be obtained by simply performing a series of 1:4 or 1:8 dilutionsafter initial 1:40 dilution. Care was taken to ensure homogeneity ofeach dilution by thorough mixing at each stage through repeatedaspiration and dispensing programmed by the precision power software.There is no dilution needed for Trolox standards and blank. FIG. 5illustrates the layout for the plate at station C. FIG. 6 shows thelayout of the 96-well plate used for a typical measurement.

Although specific features of the invention are shown in some drawingsand not in others, this is for convenience only as each feature may becombined with any or all of the other features in accordance with theinvention. The words “including”, “comprising”, “having”, and “with” asused herein are to be interpreted broadly and comprehensively and arenot limited to any physical interconnection. Moreover, any embodimentsdisclosed in the subject application are not to be taken as the onlypossible embodiments.

Other embodiments will occur to those skilled in the art and are withinthe following claims.

1. A kit for assaying the antioxidant capacity of a sample, the kitincluding: an extraction solution including a solubility enhancingcompound to be added to a sample for extracting antioxidants present inthe sample; and a fluorescent probe to be added to the extract.
 2. Thekit of claim 1 in which the extraction solution includes a high polaritysolvent and a low polarity solvent.
 3. The kit of claim 2 in which thehigh polarity solvent is water.
 4. The kit of claim 2 in which the lowpolarity solvent is selected from the group consisting of acetone,butanone, methanol, acetonitrile, methylene chloride,1-2-dichloroethane.
 5. The kit of claim 1 in which the solubilityenhancing compound is cyclodextrin and derivatives thereof.
 6. The kitof claim 2 in which the amount of the high polarity solvent is equal toor approximately equal to the amount of low polarity solvent.
 7. The kitof claim 1 in which the solubility enhancing compound is 1% to 40% ofthe solution.
 8. The kit of claim 1 in which the probe is a non-proteinprobe.
 9. The kit of claim 8 in which the non-protein probe is ahydrogen atom donor probe.
 10. The kit of claim 9 in which the hydrogenatom donor probe is fluorescein.
 11. The kit of claim 1 furtherincluding: a plurality of standards each having a known antioxidantcapacity so that the fluorescence intensity decay of the probe in thepresence of each standard over time can be detected.
 12. The kit ofclaim 11 in which there are four standards.
 13. The kit of claim 11 inwhich the concentration of the standards ranges from 10 mM to 100 mM.14. The kit of claim 11 in which each standard is Trolox.
 15. The kit ofclaim 1 further including adding a free radical generator precursor tobe added to the probe/extract mixture.
 16. The kit of claim 15 in whichthe precursor is AAPH.
 17. The kit of claim 15 in which theconcentration of the precursor is above 4 mM.
 18. The kit of claim 17 inwhich the concentration of the precursor is 12 mM.
 19. A kit forassaying the antioxidant capacity of a sample, the kit comprising: anextraction solution to be added to a sample for extracting antioxidantspresent in the solution; and a non-protein fluorescent probe to be addedto the extract.
 20. The kit of claim 19 in which the extraction solutionincludes a high polarity solvent and a low polarity solvent.
 21. The kitof claim 20 in which the high polarity solvent is water.
 22. The kit ofclaim 20 in which the low polarity solvent is selected from the groupconsisting of acetone, butanone, methanol, acetonitrile, methylenechloride, 1,2-dichloroethane, and ethanol.
 23. The kit of claim 20 inwhich the amount of the high polarity solvent is equal to orapproximately equal to the amount of low polarity solvent.
 24. The kitof claim 19 further including a solubility enhancing compound to beadded to the extraction solution.
 25. The kit of claim 24 in which thesolubility enhancing compound is cyclodextrine and derivatives thereof.26. The kit of claim 24 in which the solubility enhancing compound is 1%to 40% of the extraction solution.
 27. The kit of claim 19 in which thenon-protein probe is a hydrogen atom donor probe.
 28. The kit of claim27 in which the hydrogen atom donor probe is fluorescein.
 29. The kit ofclaim 19 further including a plurality of standards each having a knownantioxidant capacity so that the fluorescence intensity decay of theprobe in the presence of each standard over time can be detected. 30.The kit of claim 29 in which there are four standards.
 31. The kit ofclaim 30 in which the concentration of the standards ranges from 10 mMmoles to 100 mM moles.
 32. The kit of claim 29 in which each standard isTrolox.
 33. The kit of claim 19 further including a free radicalgenerator precursor to be added to the probe/extract mixture.
 34. Thekit of claim 33 in which the precursor is AAPH.
 35. The kit of claim 33in which the concentration of the precursor is above 4 mM.
 36. The kitof claim 35 in which the concentration of the precursor is 12.8.
 37. Akit for assaying the antioxidant capacity of a sample, the kitincluding: an extraction solution to be added to a sample for extractingantioxidants present in the sample; and a fluorescent probe to be addedto the extract.
 38. The kit of claim 37 in which the extraction solutionincludes a high polarity solvent and a low polarity solvent.
 39. The kitof claim 38 in which the high polarity solvent is water.
 40. The kit ofclaim 38 in which the low polarity solvent is selected from the groupconsisting of acetone, butanone, methanol, acetonitrile, methylenechloride, 1-2-dichloroethane.
 41. The kit of claim 38 in which theamount of the high polarity solvent is equal to or approximately equalto the amount of low polarity solvent.
 42. The kit of claim 37 furtherincluding: a plurality of standards each having a known antioxidantcapacity so that the fluorescence intensity decay of the probe in thepresence of each standard over time can be detected.
 43. The kit ofclaim 42 in which there are four standards.
 44. The kit of claim 42 inwhich the concentration of the standards ranges from 10 mM to 100 mM.45. The kit of claim 42 in which each standard is Trolox.
 46. The kit ofclaim 37 further including adding a free radical generator precursor tobe added to the probe/extract mixture.
 47. The kit of claim 46 in whichthe precursor is AAPH.
 48. The kit of claim 46 in which theconcentration of the precursor is above 4 mM.
 49. The kit of claim 48 inwhich the concentration of the precursor is 12 mM.